Oligopeptide prodrugs

ABSTRACT

Prodrugs are described whose structures have a oligopeptide chain which is substituted by a nucleophilic chemotherapeutic residue at the α-position. The products have increased cell membrane permeability and beneficial physico-chemical properties.

This is a divisional application of U.S. application Ser. No. 507,326,filed June 23, 1983, now U.S. Pat. No. 4,454,065, which, in turn, was acontinuation-in-part of U.S. application Ser. No. 379,537, filed May 18,1982, now abandoned.

This invention relates to oligopeptide containing prodrugs of selectedchemical compounds which are useful in the pharmaceutical art and tochemical intermediates or methods for preparing such prodrugs. Thecompounds are useful in a method for transporting impermeantchemotherapeutic agents through the cell membranes of infecting species,then, releasing the active agents intracellularly. Others, as pointedout hereafter, are also useful to prepare useful product forms or asenzyme substrates used for assaying enzyme content in biological fluids.

GENERAL BACKGROUND OF THE INVENTION

The prior art recognizes that peptide transport systems are onemechanism by which chemical substances are carried into the cellmembrane of an infecting organism. It has been postulated that bothdipeptide and oligopeptide transport systems are present in the cellmembrane, for example, in the cell membrane of Escherichia coli; B. N.Ames, Proc. Nat. Acad. Sci. OSH 70 456 (1973) or C. Gilvarg, Nature, theNew Biology 241 161 (1973). The dipeptide transport system is morespecific in its ability to transport than is the oligopeptide transportsystem.

Peptide transport systems are widespread in both procaryotic andeukaryotic microorganisms. A prodrug which can be transported per sethrough the cell membrane of infecting organisms via such a permeasesystem and, then, releases the drug within the cell would possessenhanced activity.

A number of synthetic derivatives have been prepared to take advantageof this transport system such as those described by M. M. Ponpipom, etal., J. Med. Chem. 24 1388 (1981), European Patent Office applicationNo. 38,541 or C. Philip, et al., PCT application, W0 81/01145. Some ofthese types of compounds were designed to limit toxicity or to achievemore specific biological activity. Such compounds of the prior arteither are limited in scope or are aimed at being biologically activewithout degradation at the receptor site, that is, in the transportform, due to their resistance to intracellular peptidases. For example,the cited Philip publication discloses anti-tumor moieties which arecovalently attached to a polypeptide. A covalently bound group must beactive per se. This differs from the present invention in which thewarhead or biologically active group is reversibly attached.

A number of potentially useful chemotherapeutic agents are present inthe prior art which are impermeant or poorly permeant to the cellmembrane of an infecting organism. The impermeant nature of thesecompounds may be either due to the inherent physico-chemical propertiesof the compounds or due to an acquired resistance to the drug by thepermease system of the cell membrane of the target species.

CHEMICAL BACKGROUND OF THE ART

B. Isahi, Tetrahedron (1976) 1571 or Technion, German Pat. No. 2,553,689(Farmdoc 46408X) discloses the reaction of glyoxylic acid and a primaryamide to form an α-hydroxyamino acid. It does not disclose the reactionof glyoxylic acid with an amino acid amide to form a peptide which has ahydroxy substituent at the α-carbon as described hereafter.

Publications, such as Japanese Pat. No. 54,048,773 (Farmdoc 41180B/22)and No. 53,007,619 [as well as the scientific publications correspondingto these patents, T. Nishitani, et al., J. Org. Chem. 44 2019 (1979); T.Iwasaki, et al., J. Org. Chem. 12 2419 (1977); T. Nishitani, et al.,Chem. Pharm. Bull. 28 1137 (1980)] together with R. K. Olsen, et al.,Tetrahedron Letters, 41 3579 (1975), describe the preparation of certainα-acetoxy amino acids and displacement of the α-acetoxy group bydisplacing radicals, including 5-fluorouracil or mercaptans. Nopeptides, which are reversibly α-substituted, are described in the priorart to the best of our knowledge.

DESCRIPTION OF THE INVENTION

The structures of the prodrugs of this invention have been found to havea number of requirements necessary for acceptance by the receptors ofthe permease system in the cell membrane of the infecting organisms andby the intracellular peptidases. These respective receptor systems arenecessary, first for transport and, then, for release of the active drugwithin the cell of the infecting or toxic species.

The compounds of this invention have an oligopeptide chain in which anactive moiety is reversibly substituted at the α-position of a glycylunit which, in turn, must be attached, for stabilization, to a residueof the oligopeptide chain by means of the amino group of the glycylcarrying unit.

It should be noted that, in the disclosure of this invention, the glycylcarrying unit is called the 1 unit, the stabilizing unit adjacent iscalled the 2 unit. The carboxy or C- end of the chain is represented byQ in the structures of formula I hereinafter, the amino or N- end as P.Also, one skilled in the art, reading this disclosure, will recognizethat the carrying unit may be at any position of the oligopeptide unitas long as a stabilizing amino acid is adjacent to said glycyl unit. Forconvenience of synthesis, the carrying unit is usually, and preferably,represented, herein and in the claims, as the carboxy terminal unit ofthe chain.

Therefore, oligopeptide chain will usually have a free carboxy group atthe carboxy terminal or the 1 unit, the unit to which the drug orwarhead is attached, or must be able to generate such a carboxy unit invivo. It must, even more critically, have a free amino group at theN-terminal amino acid unit or must be able to generate such a unit invivo. Each amino acid unit, other than the carrying unit, is,preferably, in its natural form. All the chemical protective groupsnecessary during the preparation of the prodrugs are removed unless theyare removed in situ in the biological system in which the end productsof this invention are to be used or serve a purpose without removal suchas with certain enzyme substrates described hereinafter.

Finally, the stereoisomeric configuration of the carrying amino acidunit must be L. The D-form has been found, to date, not to transport.The first two units of the polypeptide chain are preferably L inconfiguration. These units are the carrying unit (1 unit) and the aminocontaining (2 unit). The remaining units of the polypeptide chain areless critical and may be either D or L, or a mixture of configurations.Preferably, however, the amino acid units are all L and, for ease ofpreparation, all the same at the 3-6 units in either direction along thepeptide chain. It has been found in the dipeptide series that, when theconfiguration of the respective 1-2 units are L-L, L-DL or DL-DL, onlythe L-L form of the D-containing mixtures is transported, the compoundhaving a D-terminal unit is left behind. The two L-units of theoligopeptide at the 1 and 2 positions have, furthermore, been found tostabilize the prodrug to spontaneous decomposition.

Since the 1 unit should be L, it must be α- or 2-substituted, at leastby the "warhead" residue, that is, by the residue which is useful asdescribed hereafter. Such α-substitution, also, is important, not onlyto formation of the prodrug by a nucleophilic substitution reaction, butto release the active groups or warhead within the cells of theinfecting organism. Intracellular peptidases split the polypeptide unitbetween 1 and 2 units within the cell. The active drug is, then,released from the sole amino acid residue remaining by spontaneousdecomposition of the unit which no longer possesses the stabilizingamino acid units of the chain. In vitro evidence is presented in theutility example to support this mechanism of action. The splitting ofthe peptide chain is also important when the compounds of this inventionare used as enzyme substrates in analytical procedures.

In this disclosure, the term, oligopeptide, is used to describe thecarrying tail(s) of the prodrug but includes both dipeptide or higheroligopeptide chains. L-alanyl is preferred as the amino acid in thenon-carrying units of the peptide chain. The dipeptides are most useful.

The compounds of this invention are exemplified by the followingstructural formula: ##STR1## in which: R¹ is C₁₋₄ lower alkyl, phenyl,ω-amino-C₂₋₄ -alkyl or benzyl;

P is a hydrogen, carbobenzoxy or from 1-4 residue units of an amino acidsuch as glycyl, phenylglycyl, alanyl, phenylalanyl, lysyl, ornithyl,norvalyl, valyl, norleucyl, isoleucyl or leucyl, either in the naturalL- or in the D-configuration; and

Q is hydroxy, benzyloxy or from 1-4 residue units of an amino acid suchas glycyl, phenylglycyl, alanyl, phenylalanyl, lysyl, ornithyl,norvalyl, valyl, norleucyl, isoleucyl or leucyl, either in the naturalL- or in the D-configuration; and

W is a residue of a pharmaceutically useful agent (HW) having anelectron configuration capable of participating in a nucleophilicdisplacement reaction, said residue being reversibly substituted andbeing derived from an antimicrobial or an antiparasitic agent.

In structural formula I, the 2-peptide unit will be recognized as analanyl (or related valyl, norvalyl, leucyl, isoleucyl or norleucyl),phenylglycyl, lysyl, onithyl or phenylalanyl group, respectively.

Also, in the oligopeptide fragments, P and Q, each peptide unit may bedifferent in configuration or in chemical structure, however, forconvenience in preparation, it is preferred that the units be the sameand that they be simple in structure, fr example, poly-L-alanyl or evenpolyglycyl units.

Any reactive groups, on either an amino acid member of the peptide chainor on the warhead group, may be protected during synthesis similarly tothose referred to in the EPO reference, at page 7, line 15 and page 12,line 14. Also, see U.S. Pat. No. 3,803,120, especially at column 4, line28 as well as columns 6-9, or U.S. Pat. No. 3,957,803.

Preferred compounds of this invention are those of the followingformula: ##STR2## in which W is as defined for I above and theconfiguration is L,L.

HW, more specifically, is a compound represented by the followingstructural formula: ##STR3## in which: X is --O--, --S--, --NH--,##STR4## or activated ##STR5## and R² is the residue of apharmaceutically useful agent which is a nucleophilic attacking groupwhen taken with X.

The active agent is, more specifically, an anti-microbial oranti-parasitic agent, for example; ##STR6## (B) a C₁₋₆ -alkylthiol, afunctionalized C₁₋₆ -alkylthiol such as ω-amino-C₂₋₆ -alkylthiol, or aphenylthiol such as ##STR7## R³ is H or from 1-2 substituents of thegroup comprising C₁₋₄ -alkyl, C₁₋₄ -alkoxy, cyano, halo or C₁₋₄-alkylthio. See also CA 94 185507c, CA 93 150163q, CA 93 114552q; CA 8689671r; CA 90 98007p; CA 90 152197r; CA 92 146609b, EPO No. 38,541, U.S.Pat. Nos. 3,590,035; 3,700,676 or 3,773,770 for other antimicrobialwarheads, as well as; ##STR8## wherein R⁴ is hydrogen, halo, carboxy,trifluoromethyl, Z-bromovinyl, or nitro and V is hydrogen or carboxy, aswell as;

(D) oxibendazole, parbendazole, oxfendazole, cambendazole, oncodazole,fenbendazole, mebendazole, albendazole or thiabendazole.

Preferred compounds to be used as warhead residues or releasablemoieties in the antimicrobial or antiparasitic fields are albendazole ororotic acid derivatives.

The compounds of formula I or IA in which W is a C₁₋₆ -alkylthio, afunctionalized C₁₋₆ -alkylthio for example a C₁₋₆ -aminoalkylthio or aphenylthio as defined above by formula II are of particular utility fordetecting protease/peptidase activity in biological systems. Forexample, the compounds are more sensitive chromogenic detectors ofprotease activity in biological liquids, such as serum, than is leucinep-nitroanilide. This utility depends on the release, by protease action,of the mercaptan, H--S--R², which is assayed quantitatively bycolorimetry using Ellman's reagent as described in the examples.

L-Alanyl-L-(α-phenylthio)glycine, for example, was used as a detectorpeptide to determine protease activity in fresh and aged mouse serumwhere the use of leucine p-nitroanilide as chromogenic substrate detectslittle difference in protease level.

Detector compounds of this invention which have a free carboxy in theirstructures are used to act as chromogenic substrates forcarboxypeptidases in biological fluids.N-acetyl-L-alanyl-L-(α-phenylthio)glycine is used to monitor the actionof carboxypeptidase A using Ellman's methodology.

Detector peptides of this invention such asL-alanyl-L-(α-phenylthio)glycine have been successfully used as enzymesubstrates for leucine aminopeptidase and aminopeptidase M. Thesedetector peptides, for example, CBZAlaGly(S(CH₂)₃ NH₂)OBn, have alsobeen shown to be substrates for trypsin.

The chemical compounds of this invention are prepared by the followingreaction sequences: ##STR9##

In the reaction sequence, W, P, Q, R¹, X and R² are as described aboveor protected derivatives of the same as is conventional in the peptideart.

Z is an amino protecting agent which is used in the polypeptide art toprotect a sensitive chemical center during a reaction sequence and is,thereafter, removed by chemical means which will not affect the rest ofthe molecule, especially, in this case, the α-substituent, W. Such aminoprotective groups may be those removable either by selective hydrolysisor exchange hydrogenolysis over a noble metal, such as palladium, in thepresence of a hydrogen donor. Examples of these groups are aralkyl- oralkyloxycarbonyl groups, for example,trichloroethyloxycarbonyl,allyloxycarbonyl, benzhydryloxycarbonyl,benzyloxycarbonyl or a tert.-butyloxycarbonyl (t.-boc) type. Aprotective group which can be removed by the same chemical reaction asthe carboxyl protective group, referred to below, is preferred. See thereferences mentioned above for other protective devices known to thepeptide art.

Y is a carboxylic acid protecting group which is used for the samepurpose as Z is used for amino. Examples of such groups are the easilysplit esters such as allyl, benzhydryl, tert.-butyl, trichloroethyl,benzyl, benzyloxymethyl or p-nitrobenzyl esters.

Ac is an acyl group which, together with the oxygen to which it isattached, forms an active acyloxy leaving group known to the art.Examples of alkyl or arylcarbonyloxy groups are alkanoyl of 2-6 carbons,benzoyl, C₁₋₆ -lower alkylsulfonyl, for example, mesyl, or C₆₋₁₀-arylsulfonyl, for example, p-toluenesulfonyl. Certain leaving groups,such as the tosyloxy, may be so reactive that intermediate compoundscontaining them are not easily isolated. These may be formed and usedwithout isolation. Alternatively, a halo leaving group is used in placeof the acyloxy groups, such as an α-chloro or α-bromo group.

Examples of protective groups which are useful for synthesis of peptidesand which can be used here for Z and Y, as well as for protecting otherchemically sensitive groups in P or W, are given in Synthetic PeptidesI, Von Nostrand, 1970, pages 3-8 and in succeeding volumes in thisseries as well as in those references mentioned above.

For convenience, Z and Y are, essentially, the same protective groupswhich are both removable by a hydrogen exchange reaction over apalladium catalyst in the presence of a standard hydrogen donor.Examples of protected intermediates are those of reaction Sequence Awhich have (1) Z as carbobenzoxy and Y as benzyl, (2) Z asallyloxycarbonyl and Y as allyl, or (3) Z is tert.-butyloxycarbonyl andY is tert.-butyl. Also, when --OAc is displaced, the most practicalgroup for Ac is acetyl. A further modification is to replace the α--OHgroup in compound 3 of Sequence A with a halo leaving group, such aschloro or bromo, using halogenating agents known to the art. The haloion is, then, the leaving group in the displacement reaction, 4→6. Theα-halo intermediates are particularly of interest when the warhead groupis to be attached to the peptide chain via an oxy atom. Metallic saltsof the attacking oxy group are, then, useful.

In reaction Sequence A, the initial reaction comprises reacting aL-α-substituted amino acid amide (1) with glyoxylic acid (2) itself orwith a glyoxylate ester in which the carboxyl protective group, asdescribed above, is already in place. The reaction is carried out, atfrom room temperature to reflux, until the reaction is complete, such asfor up to 8 hours, in an inert organic solvent in which the reactantsare substantially soluble. Benzene-like solvents, refluxed over a watertrap, are most convenient. Also, halogenated solvents, such as methylenechloride, or ethereal solvents, such as ethyl ether, may also be used.

The resulting α-hydroxydipeptide (3), if not already esterified, is thenreacted with a carboxy blocking agent as known to the art.

The blocked α-hydroxydipeptide is reacted with an acylating agent suchas an acyl chloride or anhydride in the cold in a tertiary amine, suchas pyridine, to give the α-acyloxydipeptide (4).

The α-acyloxydipeptide (4) is reacted with a nucleophilic,biologically-active agent, HXR² or HW, in an inert organic solvent inwhich the reactants are soluble, at from room temperature up to refluxtemperature, until reaction is complete. A tertiary amine may be presentto take up the displaced acid. Dimethylformamide, dioxane,tetrahydrofuran or dimethylacetamide, each combined with an excess oftriethylamine, are good solvent systems.

We have found that the α-acyloxy group of the compounds of formula 4 ofreaction sequence A is very susceptible to nucleophilic displacement.The reaction favors a SN₂ course of reaction rather than a mixed SN₂-SN₁ course reported in the Tanabi references. Generally speaking, theα-acyloxy group of the compounds of formula 4 will undergo replacementmuch like that in the 3-acetoxymethyl group of certain cephalosporinintermediates, see Cephalosporins and Penicillins, E. H. Flynn, AcademicPress, (1972) pages 158-164 and 151-171.

The displacement of the α-acyloxy group of the dipeptide is a keyreaction of this invention. As stated in the discussion of the prior artabove (T. Nishitani, et al.), such displacements have been previouslyreported using an N-acyl amino acid having N-acyl groups common to theart, such as acetyl, isobutyryl or carbobenzoxy. These would not,however, serve as support moieties as do the peptide chains of the novelcompounds, described herein, which must possess the critical carryingpeptide unit in the L-configuration as well as the N-terminal aminogroup and the stabilizing 2-unit. In practice, the course of thedisplacement is followed by thin layer chromatography to ascertain theprogress of the reaction.

The important dipeptide products of the displacement (6) are thenreacted to remove any protective groups if the operator so desires, asknown to the art, such as by a catalytic exchange reaction (for example,using a palladium catalyst with a hydrogen source), acid treatment (forexample, using hydrogen chloride or hydrogen bromide in acetic acid),catalytic hydrogenation if suitable, or alkaline treatment (for example,using calcium hydroxide). Of course, such treatment must not inducechemical splitting at the critical α-warhead moiety (W). When theprotecting groups have been removed, the end compounds of this inventionof the dipeptide family (formula I, P=H) are obtained.

The oligopeptides which have more than the two amino acid units at 1 and2 are most conveniently prepared by condensing an amino acid oroligopeptide which has been N-protected as known to the art with theN-deblocked α-warhead dipeptide (Sequence A, compound 8). This step,once again, uses any peptide coupling method known to the art (see, forexample, U.S. Pat. No. 3,803,120). Examples of such methods are usingthe N-blocked amino acid and the C-blocked dipeptide withdicyclohexylcarbodiimide or carbonyldiimidazole. Other coupling methodsuse various active coupling forms of the oligo acid such as acylhalides, anhydrides, azides or active mixed esters. Once again, afterthe peptide coupling reaction, the protective groups are removed asdescribed above to obtain the final prodrugs of structural formula I.

If the peptide chain is to be extended toward the C-terminus, thedesired protected amino acid or oligopeptide unit is reacted with theprotected α-substituted oligopeptide, 11, as described immediately aboveand for Sequence B above.

As an alternative, the oligopeptide chain desired for the final productmay be in place in the structure corresponding to compound 1 of SequenceA at the glyoxalate condensation step and, then, carried through to theend product. This method, however, is not preferred since theintermediate compounds often have physico-chemical properties which makethem difficult to handle.

The products of the chemical reactions, outlined in reaction Sequences Aand B, are obtained as diastereoisomeric mixtures. These are separated,if desired by one utilizing this invention, by prior art methods such asreverse phase chromatography or fractional crystallization of either theoligopeptides (I) themselves or their salts with optically pure bases oracids. The chromatographic separation of isomers has been found,unexpectedly, a convenient resolution method. The L,L-isomers at the 1and 2 amino acid units are the ones which are selectively transported bythe peptide permease system of the cell membrane. If a mixture ofisomers is present, the L,L-isomers are absorbed selectively, leavingthe impermeable L,D-isomers behind. Therefore, either separatedL,L-isomers or diastereoisomeric mixtures are useful, with appropriateadjustments in the active quantities of the warhead moiety based on thisobservation.

The reactions, outlined above, are intended to be carried out in liquidphase, however, as with most peptide reactions, solid phase or enzymetechnology may be used alternatively, R. B. Merrifield, Biology 3 1385(1964) or J. Am. Chem. Soc. 85 2149 (1963).

Also included in this invention are the following chemical intermediateswhich have structures whose key feature is a potentially displaceablesubstituent at the α- or 2-position of the terminal glycyl unit whichis, in turn, combined with the stabilizing amino acid unit as the otherunit of the dipeptide; ##STR10## in which Y, Z and R¹ are as describedabove; and R⁵ is a leaving group as known to the chemical art innucleophilic displacement reactions, for example, chloro, bromo, hydroxyor acyloxy, with acyl being as described above.

Advantageous intermediates are those of formula III in which R¹ ismethyl, R⁵ is hydroxy or acetoxy; Z is allyloxycarbonyl, carbobenzoxy ortert.-butyloxycarbonyl, and Y is allyl, benzyl or tert.-butyl.

The prodrugs of formula I are used to enhance permeability of the cellmembrane of the target organism, against which the carriedchemotherapeutic residue (the warhead, W) is active. The compound offormula I is brought into contact with the target cells, either invitro, such as in treating a surface bacterial or fungal infection bydirect contact, or in vivo, such as in treating a systemic or localizedinfection by local infusion, parenteral injection or oraladministration.

The dose of the prodrug is derived from the known dose of the warhead ordrug itself compensated for the inactive peptide carrying chain of theprodrug. Of course, if a topical application of anantibacterial-antifungal prodrug is used, the dose is less importantthan is a sufficient excess quantity of drug applied to the site ofinfection. For topical use, from 0.5-10% concentration of prodrug isused in solution, in suspension or in a locally applied product fromsuch as in an ointment, shampoo, hair oil, troche, gum, drench or soap.

The applied prodrug, then, is transported through the cell membrane bymeans of the affinity of the peptide backbone to the receptor units ofthe permease system of the membrane. When inside the cell, the prodrugis attacked by intracellular peptidases to split the oligopeptide chain,at least between the 1 and 2-units: ##STR11## I NHCHCO₂ H + HW

in which P and W are as defined above. The drug is, then, released atthe site of action within the cells by disassociation of theα-substituted amino acid. Since the cell membrane is not easilypermeable to the active agent, the latter will tend to build up withinthe cell. Also, if the warhead (W) is a very large moiety or carries acharged group, it may not be easily transported. In this case, thenumber of peptide units in the carrying chain should be increased. Thus,the prodrug is able to use the non-specific oligopeptide transportsystem. In the case of an interferring group such as an acid group, itmight be masked reversibly or compensated for by using amino acid unitswhich are positively charged at physiological pH's, such as lysine.

While these new compounds are most useful in treating bacterial orfungal microbes topically, or even systemically in a whole animal, thesame prodrug concept can be used for other purposes. For example, thepeptide prodrug can be used to prepare injectable preparations ofcompounds not normally so used because of physico-chemical propertiessuch as low solubility. This utility may be particularly applied toknown benzimidazole anthelmintics. Of course, any route ofadministration which exposes the prodrug to peptidases prior to reachingthe site of action will destroy the peptide chain of the prodrug. Thiseffect may be, in fact, beneficial, as with certain anthelmintics (i.e.oxybendazole, albendazole, oncodazole).

The prodrugs of formula I are represented herein as their amphotericpolypeptide forms. One skilled in the art will recognize that salt formsof the prodrugs may be equally useful, such as pharmaceuticallyacceptable acid addition salts when a basic center is present in thepolypeptide chain or in the carried warhead. Alternatively,pharmaceutically acceptable basic salts derived from the usual bases,such as those having alkali metal or nontoxic organic amine cations, canbe prepared if an acid center is present. Both types of salts areprepared as known to the art, usually by contacting the prodrug with anexcess of acid or base in a suitable solvent.

Furthermore, as stated hereinabove, the compounds of formulas I and IAcan be used in the form of an amide derivative at the N-terminus such asan N-acetyl, carbobenzoxy or allyloxycarbonyl derivative or an esterderivative at the C-terminus such as an alkyl, benzyl or alkyloxyderivative.

The following examples are designed to teach the preparation of thecompounds of this invention as well as the biological activity ofrepresentative compouds of this invention. All temperatures are indegrees Centigrade.

EXAMPLE 1

A mixture of 5 g (0.0225 m) of N-carbobenzoxyalanylamide, 2.3 g (0.025m) of glyoxylic acid hydrate and 50 ml of ethyl ether-methylene chloridewas allowed to stand with stirring overnight. A white solid,N-carbobenzoxy-L-alanyl-D,L-2-hydroxyglycine, separated which weighed2.0 g, m.p. 128°-130°. The filtrate was further reacted with 2.3 g ofglyoxylic acid to obtain 3.15 g of additional product, m.p. 127°-130°.

Anal. Calcd. for C₁₃ H₁₆ N₂ O₆ : C, 52.70; H, 5.44; N, 9.46. Found: C,52.65; H, 5.06; N, 9.57.

A solution of 0.59 g (2 mm) of the α-hydroxy-dipeptide in 20 ml ofdimethylformamide was mixed with a solution of 0.33 g (1 mm) of cesiumcarbonate in 8 ml of water. The pH of the mixture was brought up to˜7.0. The volatiles were taken off with evaporation and, then,successively azeotroping with dimethylformamide and toluene. Theresulting gummy solid was dissolved in 20 ml of dimethylformamide and1.71 g (10 mm) of benzyl bromide is added. The mixture was stirred atroom temperature overnight.

The filtrate was diluted with water, then extracted with ethyl acetate.The washed extracts were combined and evaporated. The residue wastriturated under ether to give 0.49 g of white solidN-carbobenzoxy-L-alanyl-D,L-2-hydroxyglycine, benzyl ester. A sample waspurified over silica gel using 5% methanol/methylene chloride, m.p.91°-91.5°.

Anal. Calcd. for C₂₀ H₂₂ N₂ O₆ : C, 62.17; H, 5.74; N, 7.25. Found: C,62.20; H, 5.36; N, 7.04.

This reaction was also carried out using benzyl bromide, sodiumbicarbonate and dimethylformamide to give a good yield of the desiredbenzyl ester.

Acetic anhydride (3.3 ml) was added to a mixture of 0.27 g (7 mm) of theblocked α-hydroxydipeptide and 3.3 ml of pyridine cooled to 0°. Afterstanding in the cold overnight, the reaction mixture was evaporated. Theresidue was triturated with ether to give 0.32 g of whiteN-carbobenzoxy-L-alanyl-D,L-2-acetoxyglycine, benzyl ether.

Anal. Calcd. for C₂₂ H₂₄ N₂ O₇ : C, 61.67; H, 5.65; N, 6.54. Found: C,61.91; H, 5.65; N, 6.33.

The α-acetoxydipeptide (0.28 g, 6.8 mm) was reacted with 0.11 g (6.8 mm)of (benzothiazol-2-yl)methanol in 4 ml of tetrahydrofuran and 0.068 g oftriethylamine at 20°, then, stirred 24 hours. The solvent was evaporatedin vacuo to give a yellow oil,N-carbobenzoxy-L-alanyl-2-(benzothiazolyl-2-methoxy)-D,L-glycine, benzylester.

This material (150 mg) is treated with 5% palladium-on-charcoal as acatalyst and cyclohexene as a hydrogen donor in methanol to giveL-alanyl-2-(benzothiazolyl-2-methoxy)-D,L-glycine.

EXAMPLE 2

A mixture of 0.53 g (1.23 mm) of the α-acetoxy-dipeptide of Example 1,0.146 g (1.12 mm) of 5-fluorouracil, 0.113 g (1.12 mm) of triethylamineand 2 ml of dimethylformamide was stirred at room temperature for 18hours. The mixture was concentrated. The residue was dissolved in 2 mlof water, then re-extracted with ethyl acetate. The combined extractswere washed, dried and concentrated to give 0.59 g (98%) of slightlyimpure N-carbobenzoxy-L-alanyl-2-(5-fluorouracil-1-yl)-D,L-glycine,benzyl ether.

This material was purified by medium pressure liquid chromatographyusing methylene chloride/1% methanol in methylene chloride and a silicagel column to give 0.45 g (74%) of pure product as a diastereoisomericmixture, m.p. 93°-95°.

A mixture of 350 mg (0.70 mm) of the D,L-isomer mixture, 300 mg of 10%palladium-on-charcoal, 0.5 ml of cyclohexene and 35 ml of methanol washeated at reflux for 10 minutes. The hot mixture was filtered andconcentrated to give a pure deblocked product ofL-alanyl-2-(5-fluorouracil-1-yl)-D,L-glycine, 0.18 g (94%). Highpressure liquid chromatography, using a reverse phase modified silicagel C₁₈ column with an eluent of 20% methanol-water, demonstrated twodiastereoisomers in 1-1 ratio.

Anal. Calcd. for C₉ H₁₁ FN₄ O₅.H₂ O: C, 36.95; H, 4.48, N, 19.17. Found:C, 36.66, H, 4.34, N, 18.85.

The L-L and L-D diastereoisomers were separated by medium pressureliquid chromatography, after deblocking, over the silica gel columnusing the solvent system described above.

The L-D isomer had a rotation as follows: [α]_(D) ²⁵ (1, H₂ O)=-105.9°.

Anal. Calcd. for C₉ H₁₁ FN₄ O₅.H₂ O: C, 36.99, H, 4.48, N, 19.17. Found:C, 37.11; H, 4.14; N, 19.19.

The L-L isomer, had a rotation as follows: [α]_(D) ²⁵ (1, H₂ O)=+131.1°.

Anal. Calcd. for C₉ H₁₁ FN₄ O₅.1.5H₂ O: C, 35.89, H, 4.68; N, 18.59.Found: C, 35.54, 35.54; H, 4.20, 4.08; N, 18.44, 18.51.

EXAMPLE 3

Allyl glyoxylate acetal, b.p. 55°-60° at 16 mm/Hg, was prepared byreacting glyoxylic acid hydrate with an excess of allyl alcohol inbenzene.

Allyloxycarbonylamino-L-alanylamide, m.p. 135°-136°, was prepared byreacting L-alanylamide hydrochloride with one equivalent of allylchloroformate in 20 ml of water containing 2.0 g of sodium hydroxide and4 ml of tetrahydrofuran.

Anal. Calcd. for C₇ H₁₂ N₂ O₃ : C, 48.83; H, 7.03; N, 16.27. Found: C,49.16; H, 7.12; N, 16.10.

A mixture of 1.54 g (0.009 m) of theallyloxycarbonylamino-L-alanylamide, 1.2 g (0.009 m) of allylglyoxylateacetal and 50 ml of benzene was heated at reflux using azeotropicconditions for 54 hours to complete the reaction. The cooled mixture wastreated with petroleum ether and triturated to separate 2 g (80%) ofcrystalline N-allyloxycarbonyl-L-alanyl-2-hydroxy-D,L-glycine, allylester. Purification by recrystallization from ethyl acetate/petroleumether gave 1.1 g of white solid, m.p. 88°-90°.

Anal. Calcd. for C₁₂ H₁₈ N₂ O₆ : C, 50.35; H, 6.34; N, 9.88. Found: C,50.25; H, 6.55; N, 10.43.

A mixture of 0.9 g (0.003 m) of the α-hydroxy-dipeptide, 7.0 ml ofacetic anhydride and 9 ml of dry pyridine was prepared in the cold.After standing 8 hours, the volatiles were taken off in vacuo to leave0.85 g of yellow oil. Thin layer analysis showed some starting materialpresent which was separated by trituration with ether to giveN-allyloxycarbonyl-L-alanyl-2-acetoxy-D,L-glycine, allyl ester. NMR(CDCl₃), δ, 1.35 (d,3); 2.0 (s,3); 4.5 (t,2) 5-6 (m³, olefinics); 6.4(d,2).

A mixture of 0.75 g (0.0022 m) of the α-acetoxy-dipeptide, 0.25 g(0.0022 m) of benzothiazol-2-ylmethanol and 10 ml of dry tetrahydrofuranwas added to 0.22 g (0.0022 m) of dry triethylamine in 5 ml of drytetrahydrofuran. The mixture was stirred at room temperature for 8hours. Thin layer chromatography (99:1, methylene chloride:methanol)showed the reaction was complete. The mixture was evaporated in vacuo togive 1.1 g of yellow oil, which was purified by medium pressure liquidchromatography over a silica gel column (2% methanol in methylenechloride), to give 0.51 g (54%) ofN-allyloxy-carbonyl-L-alanyl-2-(benzothiazolyl-2-methoxy)-D,L-glycine,allyl ester. NMR (CDCl₃), δ, 1.4 (d,3); 4.55 (t,2); 5.15 (s,2); 5.2-6.2(m³, olefinics); 7.4 and 8.0 (m, aromatics).

A mixture of 0.3 g (0.0007 m) of the displacement product and 3 ml ofdry methylene chloride under nitrogen was treated with a solution of 0.5g of 2-ethylhexanoic acid, as hydrogen donor, in 3 ml of methylenechloride, followed by 40 mg of triphenylphosphine and 40 mg oftetra(triphenylphosphine)palladium. The mixture was stirred at roomtemperature overnight, then, diluted with ether. The resulting solid (18mg) was collected.

The filtrate was concentrated to leave a residue which was largelystarting material.

This material was reacted again, as above, for 24 hours, to give 85 mgof deblocked product, to give a total yield of 103 mg ofL-alanyl-2-(benzothiazolyl-2-methoxy)-D,L-glycine as the zwitterion, RF0.47 (80:20 acetonitrile/water).

Anal. Calcd. for C₁₃ H₁₅ N₃ O₄ S: C, 50.48; H, 4.89; N, 13.58. Found: C,50.70; H, 4.84; N, 13.40.

EXAMPLE 4

A mixture of 0.28 g (0.001 m) of4-(N-(2-mercaptoethyl)amino)-pyridine-2,6-dicarboxylic acid, 0.33 g(0.001 m) of the blocked α-acetoxydipeptide from Example 3, 0.44 g(0.004 m) of dry triethylamine and 13 ml of dry dimethylformamide wasstirred at room temperature for two hours. Some unreacted amine salt ofthe cysteamine starting material was separated. The filtrate wasevaporated to leave a residue which was dissolved in water, acidified topH 1.5 and allowed to evaporate overnight. A solid, 105 mg, separatedwhich was the desired free base,N-allyloxycarbonyl-L-alanyl-2-(2,6-dicarboxy-pyridyl-4-)-ethylthio-D,L-glycine,allyl ester. NMR (DMSO-d₆), δ, 1.2 (d,3); 2.85 (m,2); 3.5 (m,2); 4.2(t,1); 4.5 (m,2); 5.1-6.1 (m³, olefinic); 7.4 (s,2).

Anal. Calcd. for C₂₁ H₂₆ N₄ O₉ S: C, 48.63; H, 4.97; N, 10.65. Found: C,49.30; H, 5.13; N, 10.97.

A mixture of 220 mg (0.0004 m) of the blocked cysteamine displacementproduct, 0.09 ml of triethylamine and 10 ml of dry methylene chloridewas reacted with 0.66 g of 2-ethylhexanoic acid in 3 ml of dry ethylacetate, followed by 20 mg each of triphenylphosphine andtetra(triphenylphosphine)palladium. The mixture was stirred overnight.The separated white solid was collected, washed with methylene chlorideand dried to give 150 mg (97%) ofL-alanyl-2-(2,6-dicarboxy-pyridyl-4-)-ethylthio-D,L-glycine as themonotriethylamine salt; U.V. Emax=11,000 at 300 wave lengths in water.

Anal. Calcd. for C₁₄ H₁₈ N₄ O₇ S.N(C₂ H₅)₃ : C, 49.27; H, 6.82; N,14.37. Found: C, 49.13; H, 6.80; N, 14.71.

EXAMPLE 5

A mixture of 0.175 g (0.64 mm) ofL-alanyl-2-(5-fluorouracil-1-yl)-D,L-glycine hydrate, prepared as above,0.1089 of sodium bicarbonate and 2.5 ml of water was combined with 0.204g (1.28 mm) of N-carbobenzoxy-L-alanine, N-hydroxysuccinimide ester,prepared from the blocked alanine and N-hydroxysuccinimide carbonate,dissolved in 3 ml of dioxane. The mixture was stirred at roomtemperature for 5 hours, then concentrated. The residue was diluted with7 ml of water and 7 ml of ethyl acetate, then, adjusted to pH 3.2 with10% citric acid solution. The organic layer was separated and combinedwith ethyl acetate washings of the aqueous layer, then, washed with 10%citric acid solution. The dried organic extract was evaporated to give0.15 g (50%) of a white solid, which isN-carbobenzoxy-L-alanyl-L-alanyl-2-(5-fluorouracil-1-yl)-D,L-glycine.

This material (0.15 g) was dissolved in 15 ml of methanol and heated atreflux with 0.15 g of palladium-on-charcoal and 0.5 ml of cyclohexanefor 10 minutes. The hot reaction mixture was filtered, concentrated,redissolved in water and lyophilized. The product was, then, purified ona C-18 silica gel reverse phase medium pressure liquid chromatographiccolumn (H₂ O) to give, after lyophilizing,L-alanyl-L-alanyl-2-(5-fluorouracil-1-yl)-D,L-glycine.

Anal. Calcd. for C₁₂ H₁₆ N₅ O₆.2H₂ O: C, 37.80; H, 5.28; N, 18.36.Found: C, 37.98; H, 4.78; N, 18.05.

EXAMPLE 6

A mixture of 0.328 g (1.0 mm) ofN-allyloxy-carbonyl-L-alanyl-2-acetoxy-D,L-glycine, allyl ester and0.249 g (1.0 mm) of oxibendazole (methyl5-n-propoxy-2-benzimidazolecarbamate), 0.10 g of triethylamine and 5 mlof dimethylformamide is stirred at room temperature for 12 hours. To theresulting mixture was added 50 ml of ethyl acetate followed with 50 mlof 1.5% hydrochloric acid. The ethyl acetate fraction was separated andwashed with water and brine in succession. The dried extract wasconcentrated to give 0.305 g (59%) ofN-allyloxycarbonyl-L-alanyl-2-(5-n-propoxy-2-carbomethoxyaminobenzimidazol-1-yl)-D,L-glycine,allyl ester. Thin layer chromatography over silica gel (95:5 methylenechloride/methanol) shows one major product. This material was furtherpurified using the medium pressure liquid chromatography over silicagel, using methylene chloride to 2% methanol in methylene chloride togive 0.166 g of purified product.

This blocked product (0.157 g, 0.30 mm) was combined with 50 mg oftetra(triphenylphosphine) palladium, 10 mg of triphenylphosphine and 5ml of ethyl acetate as above. The mixture was stirred under nitrogen for3 hours. The separated product (50 mg) was purified by recrystallizationfrom aqueous acetone to giveL-alanyl-2-(5-n-propoxy-2-carbomethoxyaminobenzimidazol-1-yl-D,L-glycine.The compound demonstrated two ultraviolet and ninhydrin positive spotson thin layer, indicative of the two diastereoisomers.

This compound, 10 mg/kg of warhead equivalent, is dissolved in salineand administered by subcutaneous injection to a swine or horse infectedwith a helminth infestation susceptible to oxibendazole.

Other benzimidazoles having a reactive hydrogen at the 1-position, aslisted above, may be prepared and used similarly. The dipeptide chain(I, n=o) is preferred for injectable use because of the good solubilityof the resulting prodrug.

EXAMPLE 7

A mixture of 0.214 g (0.05 mm) of the blocked 2-acetoxyglycine ofExample 1 and 2 ml of dry tetrahydrofuran was mixed with a solution 0.11g (0.1 mm) of 2-mercaptopyridine, 0.119 (0.1 mm) of triethylamine and 5ml of tetrahydrofuran. The latter solution was added dropwise, over a 20minute period, to the acetoxy mixture. The reaction mixture was stirredat ambient temperature overnight.

The solvents were combined and evaporated to give a yellow oil which wastaken up in ethyl acetate, washed with water and brine, then dried. Theextract was evaporated to leave a yellow oil. NMR spectrum analysisshowed the desired displacement product with a trace of startingacetate. Liquid chromatography using 5% methanol:methylene chloridedemonstrated the separation of isomers.

N-Carbobenzoxy-L-alanyl-2-(pyridyl-2-thio)-D,L-glycine, benzyl ester(200 mg) was dissolved in glacial acetic acid and dry hydrogen chloridegas was bubbled through the mixture for 4 hours. Thin layer demonstratedonly partial splitting. Hydrogen bromide gas was bubbled through themixture. An exothermic reaction occurred in the mixture which was cooledin an ice bath. The blocked product disappeared quickly as analyzed bythin layer chromatography (cellulose plate, 4:1:4, butanol/aceticacid/water).

The mixture was concentrated and triturated with ether to give 0.132 gof L-alanyl-2-(pyridyl-2-thio)-D,L-glycine. This material containedminor quantities of impurities so it was passed through C-18 reversephase medium pressure liquid chromatographic purification.

This same procedure is used using 2-mercaptopyridone as the displacingwarhead to give D,L-alanyl-2-(1-oxypyridyl-2-thio)-D,L-glycine.

EXAMPLE 8

A mixture of 12.2 g (0.055 m) of carbobenzoxyalanylamide, 10 g (0.061 m)of benzyl glyoxalate and 150 ml of toluene was heated under vacuum atabout 70° overnight, allowing the mixture to reflux over a water trap.The toluene was evaporated from the reaction mixture to give a whitesolid. This was treated with ethyl acetate. The dried extract was cooledto give 12.6 g of N-carbobenzoxy-L-alanyl-D,L-2-hydroxyglycine, benzylester identical to the intermediate characterized in Example 1.

This compound (500 mg) is reacted with 1-methyltetrazol-5-thiol or1-(2-methanesulfonamidoethyl)tetrazol-5-thiol followed by acid treatmentas described above to giveD,L-alanyl-2-(1-methyltetrazolyl-5-thio)-D,L-glycine andD,L-alanyl-2-(1-(2-methanesulfonamido)ethyltetrazolyl-5-thio)-D,L-glycine.

EXAMPLE 9

A mixture of 0.386 g of N-carbobenzoxy-L-alanyl-2-hydroxy-D,L-glycine,benzyl ester, from Example 1 and 10 ml of thionyl chloride was stirredat room temperature overnight, then concentrated. The residue wastriturated with petroleum ether to give, after filtration and drying,0.364 g of N-carbobenzoxy-L-alanyl-2-chloro-D,L-glycine, benzyl ester.

Anal. Calcd. for C₂₀ H₂₁ ClN₂ O₅ : C, 59.33; H, 5.23; N, 6.92; Cl, 8.76.Found: C, 59.20; H, 5.38; N, 7.08; Cl, 8.68. The NMR spectrum showed aproton peak at 6.28 ppm. The α-hydroxy peak is at 5.55 ppm.

The halogenation was repeated using dimethoxyethane as solvent and areflux period overnight. After thin layer analysis, the reaction mixturewas combined with 35.2 mg of the disilver salt of(-)-(cis-1,2-epoxypropyl)phosphonic acid (prepared from the sodium saltin water, U.S. Pat. No. 3,929,840). The mixture was stirred overnight,then filtered and concentrated to give an oil. The product was dissolvedin ethyl acetate, washed with water, dried and concentrated to leave29.4 g ofN-carbobenzoxy-L-alanyl-2-(-)-(cis-1,2-epoxypropyl)-phosphono-D,L-glycine,benzyl ether which is a compound with phosphonomycin as the warhead.

This compound is deblocked using exchange hydrogenation as describedabove.

EXAMPLE 10

A mixture of 1.2 g (0.0036 m) ofN-allyloxy-carbonyl-L-alanyl-2-acetoxy-D,L-glycine, allyl ester,prepared as in Example 3, and 15 ml of dry dimethylformamide was mixedwith 0.5 g (0.0036 m) of p-nitroaniline and 0.36 g (0.0036 m) of drytriethylamine. The mixture was stirred at room temperature for 67 hours,then poured into 300 ml of ice-water and stirred. The precipitatedyellow solid was collected, washed with water and dried to give 1.22 g(83.5%) of N-allyloxycarbonyl-L-alanyl-2-(p-nitroanilino)-D,L-glycine,allyl ester. The product is recrystallized from hot methylene chloride,m.p. 160°-161°.

Anal. Calcd. for C₁₈ H₂₂ N₄ O₇ : C, 53.70; H, 5.46; N, 13.79. Found: C,52.92; H, 5.56; N, 13.72.

A mixture of 0.366 g (0.0009 m) of the displacement product and 10 ml ofmethylene chloride was mixed with a mixture of 0.33 g of 2-ethylhexanoicacid and 10 ml of ethyl acetate followed by 0.04 g of triphenylphosphineand 0.04 g of palladium tetra(triphenylphosphine). After stirring for 20hours, the mixture was worked up as described above to give 30 mg ofL-alanyl-2-(p-nitroanilino)-D,L-glycine, m.p. 174°-175° (dec.).

Anal. Calcd. for C₁₁ H₁₄ N₄ O₅ : C, 46.81; H, 5.00; N, 19.85. Found: C,48.73; H, 5.41; N, 18.65.

EXAMPLE 11

The α-acetoxypeptide used in Example 10, 0.66 g, was reacted with 0.28 gof p-nitrophenol as described to give 0.16 g of a white solid,N-allyloxycarbonyl-L-alanyl-2-(p-nitrophenoxy)-D,L-glycine, allyl ester,m.p. 121°-122°.

Deblocking using the hydrogenation exchange procedure of Example 10 gaveL-alanyl-2-(p-nitrophenoxy)-D,L-glycine, m.p. 105°-108°, slightlycontaminated with unblocked starting material.

EXAMPLE 12

A mixture of 0.26 g (0.001 m) of5-(hydrazinosulfonyl)-1,3-benzenedicarboxylic acid, 0.43 g (0.001 m) ofN-carbobenzoxy-L-alanyl-2-acetoxy-D,L-glycine, benzyl ester and 5 ml ofdry dimethylformamide is mixed with 0.3 g (0.003 m) of triethylamine.The mixture was stirred at room temperature for 4 hours. Thin layer(cellulose, 97:3:3-methylene chloride:methanol:formic acid) demonstratedstarting materials were gone with a new spot present.

The volatiles were removed at the pump. The residue was treated withdilute hydrochloric acid. The separated oil was taken up in ethylacetate. The combined extracts were dried and evaporated in vacuo togive a glassy residue ofN-carbobenzoxy-L-alanyl-2-(3,5-dicarboxyphenylsulfohydrazino)-D,L-glycine,benzyl ester which could not be crystallized but had a consistant NMRcurve.

This material (0.54 g, 0.0008 m) in 300 ml of methanol was reacted with1.0 ml of cyclohexene, 0.54 g of 10% palladium-on-charcoal at reflux for1 hour. Working up as described above gave product which was not activein the biological screen described in the utility example but theproduct on further analysis was found to be not completely deblocked.

The material was deblocked as noted in Example 2, using a longerreaction time, to give the desiredL-alanyl-2-(3,5-dicarboxy-phenylsulfohydrazino)-D,L-glycine.

EXAMPLE 13

A mixture of 2.14 g (5 mm) ofN-carbobenzoxy-L-alanyl-D,L-2-acetoxyglycine benzyl ester, 0.55 g (5 mm)of thiophenol, 0.5 g (5 mm) of triethylamine and 25 ml ofdimethylformamide was stirred at room temperature for 18 hours. Themixture was concentrated. The residue was dissolved in ethyl acetate.The organic extract was washed with water (3×50 ml), dried (MgSO₄),concentrated and flash chromatographed over silica gel to give 1.6 g(67%) of analytically pure oilyN-carbobenzoxy-L-alanyl-2-(thiophenyl)-D,L-glycine benzyl ester as amixture of diasteriomers.

A mixture of 1.4 g (2.9 mm) of blocked peptide prepared above wasdissolved in 10 ml of glacial acetic acid and cooled to 0°. A 25 mlsaturated solution of glacial acetic acid and hydrobromic acid was addedto the cooled acetic acid solution of blocked peptide. The mixture wasstirred for 30 minutes. The cooling bath was, then, removed and thesolution stirred for 5 hours. Solvents were evaporated to produce aviscous solid (gum) which solidified on trituration with methylenechloride to give 0.4 g (54%) of a hygroscopic HBr salt. This solid saltwas converted to the zwitterion by dissolving in ethanol and treatingthe resulting solution with 5 ml of propylene oxide which, after 5hours, produced 0.2 g of a white solid,L-alanyl-2-(thiophenyl)-D,L-glycine.

High pressure liquid chromatography (HPLC) using a reverse phasemodified silica gel C₁₈ column with a stepped eluent of 100% water (300ml), 10% methanol-water (1 l), 20% methanol-water (1.5 l), separated theL-L and the L-D diastereoisomers.

The L-L isomer had a rotation as follows: [α]_(D) ²⁵ (1, H₂ O)=+170.0.

Anal. Calcd. for C₁₁ H₁₄ N₂ O₃ S.1/4H₂ O: C, 51.05; H, 5.64; N, 10.82.Found: C, 51.37; H, 6.65; N, 10.42.

The L-D isomer had a rotation as follows: [α]_(D) ²⁵ (1, H₂ O)=-200.8.

Anal. Calcd. for C₁₁ H₁₄ N₂ O₃ S.1/4H₂ O: C, 51.05, H, 5.64, N, 10.82.Found: C, 51.11; 5.65; N, 10.25.

EXAMPLE 14

The α-acetoxypeptide, which was used in Example 13, (5 mm) is reactedwith 2,4-dichlorothiophenol (5 mm) using triethylamine (5 mm) in 25 mlof dimethylformamide to give the desired blocked peptide.

The carbobenzoxy and benzyl blocking groups are removed as previouslydescribed using hydrobromide acid, acetic acid to give the hydrobromidesalt of the desired peptide which was converted to the zwitterion bytreatment with propylene oxide. The isomers ofL-alanyl-2-(2,4-dichlorothiophenyl)-D,L-glycine are separated asdescribed in Example 13.

EXAMPLE 15

The α-acetoxypeptide from Example 13, (5 mm), 4-acetamidothiophenol (5mm), triethylamine (5 mm) and 25 ml of dimethylformamide are reacted andresulting blocked peptide deblocked as described. The resultingdiasteriomeric mixture is separated using high pressure liquidchromatography as described to giveL-alanyl-2-(4-acetamidothiophenyl)-D,L-glycine.

EXAMPLE 16

The α-acetoxypeptide (5 mm), ethylmercaptan (5 mm), triethylamine (5 mm)and 25 ml of dimethylformamide are reacted as previously described. Theresulting blocked peptide is treated with hydrobromic acid in aceticacid as described to give the hydrobromide ofL-alanyl-2-(thioethyl)-D,L-glycine which is converted to the zwitterionby treatment with propylene oxide. The D,L-diastereomeric mixture isseparated as described in Example 13.

L-Alanyl-2-(thiobutyl)-D,L-glycine is prepared using butyl mercaptan (5mm). L-alanyl-2-(thiobenzyl)-D,L-glycine is prepared usingbenzylmercaptan (5 mm). L-Alanyl-2-(3-aminopropylthio)-D,L-glycine isprepared using N-carbobenzoxy-3-aminopropylmercaptan (5 mm).

EXAMPLE 17

A mixture of 4.28 g (10 mm) ofN-carbobenzoxy-L-alanyl-D,L-2-acetoxyglycine benzyl ester, 0.94 g (10mm) of phenol, 10 g of (10 mm) triethylamine and 35 ml ofdimethylformamide was stirred at room temperature for 18 hours. Themixture was concentrated. The residue was dissolved in ethyl acetate,washed with water (3×100 ml), dried (MgSO₄) and evaporated to giveimpure L-alanyl-2-(phenoxy)-D,L-glycine as the ester derivative.Purification was obtained by using medium pressure chromatography over asilica gel column with an eluent of methylene chloride (300 ml) and 10%methanol-methylene chloride (1 l).

A mixture of 0.75 g (1.5 mm) of the D,L-isomeric mixture, purified asdescribed in Example 13, was dissolved in 50 ml of methanol with heatingand 0.4 g of 10% palladium-on-charcoal was added. The mixture washydrogenated using a Parr hydrogenation apparatus for a total of 8.5hours. The catalyst was filtered off. The filtrate was evaporated. Theresidue was dissolved in water, filtered and lyophylized to give 0.26 g(73%) of a diastereoisomeric mixture ofL-alanyl-2-(phenoxy)-D,L-glycine.

High pressure liquid chromatography (HPLC), using a reverse phasemodified silica gel C₁₈ column with an eluent of 100% water (300 ml),10% methanol-water (1 l), 20% methanol-water (1 l), separated the L-Land the L-D isomers.

The L-L isomer had a rotation as follows: [α]_(D) ²⁵ (1, H₂ O)=-61.8.

Anal. Calcd. for C₁₁ H₁₄ N₂ O₄.3/4H₂ O: C, 52.47; H, 5.96; N, 11.13.Found: C, 52.76; H, 5.77; N, 11.30.

The L-D isomer had a rotation as follows: [α]_(D) ²⁵ (1, H₂ O)=+54.9.

Anal. Calcd. for C₁₁ H₁₄ N₂ O₄.3/4H₂ O: C, 52.47; H, 5.96; N, 11.13.Found: C, 52.72; H, 5.62; N, 11.10.

L-Alanyl-2-(2,4-dichlorophenoxy)-D,L-Glycine

As above using 2,4-dichlorophenol 15 mml.

L-Alanyl-2-[4-(2-aminoethyl)phenoxy]-D,L-Glycine

As above using 4-(N-carbobenzoxy-2-aminoethyl)phenol.

EXAMPLE 18

A mixture of 7.63 g (35.5 mm) alanine benzyl ester hydrochloride, 4.08 g(35.5 mm) of N-ethylmorpholine and 150 ml of dimethylformamide wascooled to 0°. To this mixture was added 10.5 g (35.5 mm) ofN-carbobenzoxy-L-alanyl-D,L-2-hydroxyglycine, 4.79 g (35.5 mm) ofhydroxybenzotriazole and 8.04 g (39.1 mm) of dicyclohexylcarbodiimide.The reaction mixture was stirred at 0° for 45 minutes and, then, for 15hours at room temperature after which the crude reaction mixture wasfiltered and concentrated. The residue was dissolved in 500 ml of ethylacetate, washed with dilute sodium bicarbonate solution (3×100 ml), 10%citric acid solution (3×100 ml), water (3×100 ml), saturated sodiumchloride solution (3×100 ml) and dried (MgSO₄). Evaporation of thesolvent produced a white solid which was recrystallized from anacetone-hexane mixture (2:1) to give 16.3 g (100%) ofN-carbobenzoxy-L-alanyl-D,L-2-hydroxyglycyl-L-alanine benzyl ester.

A 1.0 g (2.9 mm) portion ofN-carbobenzoxy-L-alanyl-D,L-2-hydroxyglycyl-1-alanine benzyl ester wastreated with 25 ml of acetic anhydride and cooled to ˜0° in an ice-waterbath. Then, 25 ml of pyridine was added and the reaction mixture storedat 5° for 15 hours. The reaction mixture was concentrated. The resultingresidue was triturated with a 1:1 ethyl ether-petroleum ether solution,filtered and dried to give 0.82 g (75%) ofN-carbobenzoxy-L-alanyl-D,L-2-acetoxyglycyl-L-alanine benzyl ester (TLC95:5 methylene chloride methanol, silica gel, R_(f) =0.64; ¹ H and ¹³ CNMR consistent with structure.)

A mixture of 2.0 g (4.0 mm) ofN-carbobenzoxy-L-alanyl-D,L-2-acetoxyglycyl-L-alanine benzyl ester, 3.5g (12 mm) of trimethioprim and 50 ml of dimethylformamide was stirredfor 1 hour. The mixture was concentrated. The residue was dissolved in400 ml of ethyl acetate and washed with water (3×75 ml) and brine (75ml), dried (MgSO₄), concentrated. The residue was flash chromatographedto give 1.95 g (67%) of a foamy solid,N-carbobenzoxy-L-alanyl-2-[[4-amino-5[(3,4,5-trimethoxyphenyl)methyl]pyrimidin-2-yl]-amino]D,L-glycyl-L-alaninebenzyl ester, which was characterized by nuclear magnetic resonance(NMR) and mass spectral analysis.

A mixture of 0.76 g (1.04 mol) of the blocked peptide, 0.37 g of 10%palladium-on-charcoal and 100 ml of methanol was hydrogenated in a Parrapparatus at 50 psi for 14 hours. The catalyst was filtered off. Thefiltrate was concentrated, dissolved in water and lyophilized to give0.48 g ofL-alanyl-2-[[4-amino-5[(3,4,5-trimethoxyphenyl)methyl]-pyrimidine-2-yl]amino]-D,L-glycyl-L-alanine.Rapid medium pressure chromatography gave the analytically pure mixtureof diastereoisomers. Careful chromatography on the same mixturesuccessfully separated the diastereomers.

The L,L,L isomer had a rotation as follows: [α]_(D) ²⁵ (1, H₂ O)=-27.0.

Anal. Calcd. for C₂₂ H₃₁ N₇ O₇.1-3/4H₂ O: C, 49.20; H, 6.47; N, 18.25.Found: C, 49.10; H, 6.33; N, 18.16.

The LDL isomer had a rotation as follows: [α]_(D) ²⁵ (1, H₂ O)=-58.0.

Anal. Calcd. for C₂₂ H₃₁ N₇ O₇.2H₂ O: C, 48.79; H, 6.51; N, 18.10.Found: C, 48.54; H, 6.27; N, 18.24.

EXAMPLE 20

A mixture of 2.14 g (5.0 mm) ofN-carbobenzoxy-L-alanyl-D,L-2-acetoxyglycine benzyl ester, 0.46 g (5.0mmol) of aniline, 0.50 g (5.0 mm) of triethylamine and 50 ml ofdimethylformamide was stirred at room temperature for 2 hours. Themixture was concentrated. The residue was dissolved in ethyl acetate andwashed with water (3×50 ml) and brine (50 ml), dried (MgSO₄),concentrated and flash chromatographed over silica gel to give 2.46 g(100%) of N-carbobenzoxy-L-alanyl-2-(anilinyl)-D,L-glycine benzyl ester.Crystallization from hexane/acetone gave an analytically pure sample asa mixture of diastereomers.

A mixture of 0.81 g (1.75 mmol) of the blocked peptide prepared above,0.4 g of 10% palladium-on-charcoal and 150 ml of methanol washydrogenated in a parr apparatus at 50 psi for 8 hours. The catalyst wasfiltered. The filtrate was concentrated, dissolved in water andlyophilized to give 0.40 g of a mixture of diastereomericL-alanyl-2-(anilino)-D,L-glycines.

A 200 mg portion of this material was separated into the purifieddiastereomers by medium pressure reverse phase chromatography asdescribed above.

The L,L isomer had a rotation as follows: [α]_(D) ²⁵ (1, H₂ O)=+70.0.

Anal. Calcd. for C₁₁ H₁₅ N₃ O₃.1-1/2H₂ O: C, 49.99; H, 6.86; N, 15.96.Found: C, 49.97; H, 6.51; N, 15.96.

The L,D isomer had a rotation as follows: [α]_(D) ²⁵ (1, H₂ O)=+39.9.

Anal. Calcd. for C₁₁ H₁₅ N₃ O₃.1-1/2H₂ O: C, 49.99; H, 6.86; N, 15.96.Found: C, 49.93; H, 6.51; N, 16.06.

EXAMPLE 21

A 2.5 g (5.0 mmol) portion ofN-carbobenzoxy-L-alanyl-α-acetoxyglycyl-L-alanine benzyl ester, 0.51 ml(0.55 g, 5.0 mmol) of thiophenol and 0.51 g (5 mmol) of triethylaminewere combined in 50 ml of dimethyl formamide. Thin layer chromatography(TLC) (95:5 methylene chloride, methanol, silica plates) indicated thedisappearance of acetate after 0.25 hours. After 30 minutes, thereaction was concentrated, dissolved in 200 ml of ethyl acetate andwashed with 3×25 ml of water and 25 ml of brine, dried (MgSO₄) andconcentrated to give 2.31 g of a light yellow foam,N-carbobenzoxy-L-alanyl-D,L-(α-S-phenyl)glycyl-L-alanine benzyl ester.TLC indicated minor impurities.

The material was flash chromatographed 1.5"×6" column of Baker flashsilica gel, eluting with methylene chloride followed by 29% methanol inmethylene chloride to give 2.25 g (82%) of pure product. ¹ HNMR (CDCl₃)δ8.0-7.0 (m, 15), 6.2-58 (m, 2), 5.25 (S, 1), 5.15 (S, 1), 5.10 (S, 2),4.8-4.2 (m, 3), 1.3 (t, 6); ¹³ C NMR (CDCl₃) δ172.2, 171.8, 166.9,155.8, 136.3, 135.3, 134.8, 130.1, 128.8, 128.5, 128.3, 128.0, 127.8,67.0, 66.7, 57.6, 50.4, 48.4, 18.9, 18.2, 18.0. H(CH₂ Cl₂) 3420, 3040,2990, 1740, 1660, 1500 cm⁻¹.

A 1.0 g (1.8 mmol) portion of the blocked diastereomeric mixture wasadded to 40 ml of hydrobromic acid saturated acetic acid at 20°. Thereaction was stirred at room temperature for 6 hours. The volatiles wereremoved under vacuum. The resulting oil was triturated with ether,filtered, dissolved in 10 ml of ethanol and treated with an excess (3ml) of propylene oxide, then, refrigerated for 16 hours.

The resulting solid was filtered, washed with acetone and dried to give0.42 g (71%) of the zwitterion.

The diastereomeric mixture was injected onto a RP-18 MPLC column, whichwas described above, and eluted with a stepwise gradient of 0.5 L ofwater and 1 L of 20% methanol in water to elute the more mobileL-alanyl-L-(S-phenyl)-glycyl-L-alanine; 76 mg (18%) and, then, the lessmobile D-isomer.

The L,L,L isomer had a rotation as follows: [α]_(D) ²⁵ (1, H₂ O)=+102.9.

Anal. Calcd. for C₁₄ N₁₉ N₃ O₄ S.1-1/4H₂ O: C, 48.33; H, 6.23; N, 12.07.Found: C, 48.06; H, 5.81; N, 12.56.

The L,D,L isomer had a rotation as follows: [α]_(D) ²⁵ (1, H₂ O)=-150.6.

Anal. Calcd. for C₁₄ H₁₉ N₃ O₃ S.1-1/8H₂ O: C, 48.64; H, 6.20; N, 12.15.Found: C, 48.08; H, 5.84; N, 12.72.

EXAMPLE 22

A mixture of 0.428 g (1.0 mmol) ofN-carbobenzoxy-L-alanyl-D,L-2-acetoxyglycine benzyl ester, 0.265 g (1.0mmol) of albendazole, 0.101 g (5.0 mmol) of triethylamine and 10 ml ofdimethylformamide was stirred at room temperature for 6 hours. Theresulting solution was concentrated. The residue was dissolved in ethylacetate, washed with water (3×50 ml) and brine (50 ml), dried (MgSO₄),concentrated and redissolved in methylene chloride. A minor amount ofinsoluble material was filtered off and the filtrate was flashchromatographed (MPLC) over silica gel to give 0.25 g (40%) of a whitefoamy solid. Spectroscopic analysis (¹ N and ^(u) C nmr, ir) wasconsistant with a mixture of isomers ofN-carbobenzoxy-L-alanyl-2-(albendazole)-D,L-glycine benzyl ester. HPLCalso indicated a mixture.

A 0.80 g (1.26 mmol) portion of blocked peptide mixture was added to 50ml of acetic acid saturated with hydrobromic acid and then stirred atroom temperature for 6 hours. The reaction was poured into 1 L of ether,filtered and dried under vacuum. The resulting solid HBr salt wasdissolved in 20 ml of absolute ethanol and combined with 2 ml ofpropylene oxide. The white solid which formed on slow cooling to -4° wasfiltered off and dried under vacuum to give 0.217 g (53%) ofL-alanyl-2-(albendazole)-D,L-glycine.

Anal. Calcd. for C₁₇ H₂₃ N₅ O₅ S.1/2H₂ O: C, 48.79; H, 5.78; N, 16.73.Found: C, 48.46; H, 5.54; N, 16.22.

This compound was found to have antiparastic activity comparable to, orgreater than, albendazole against Fasciola hepatica and Nematospiroidesdubius infections in mice given subcutaneously at 15 mg/kg for eightdays. In an in vitro screen against Caenorhabditis elegans, nematostaticactivity was demonstrated at concentrations of 10 and 100 μg/ml.

EXAMPLE 23

A 1.68 g (20 mmol) portion of sodium bicarbonate in 120 ml of 1:1tetrahydrofuran-water was deoxygenated with a stream of argon gas.Aminopropylthiol hydrochloride (1.27 g, 10 mmol) was added followed by2.35 g (10.8 mmol) of di-t-boc carbonate. The mixture was heated atreflux for 2 hours maintaining argon flow. The cooled mixture wasneutralized to pH 7 and concentrated. The aqueous phase was acidified topH 2 and extracted with 2×75 ml portions of ethyl acetate. The combinedextracts were washed with 1.5N hydrochloric acid and brine. The driedand washed extract was concentrated to an oil which was pumped under ahigh vacuum overnight. A total of 1.8 g of material which was largelythe t-boc derivative by thin layer analysis (95:5 methylenechloride-methanol).

The crude sample was purified by flash chromatography to give 1.61 g ofpure N-t-boc-3-aminopropylthiol.

A mixture of 0.428 g (1.0 mmol) of the 2-acetoxy starting materialprepared as described in Example 1, 0.191 g (1.0 mmol) of the t-bocprepared above, 0.1 g (1 mmol) of triethylamine and 1.0 ml ofdimethylformamide was stirred for 12 hours. The mixture was concentratedand partitioned between 25 ml of water and 2×35 ml of ethyl acetate. Theorganic extract was washed with water and brine, dried and concentratedto give 0.66 g of the α-thiol t-boc product. The product was flashchromatographed, then, further purified by medium pressure liquidchromatography using 1% methanol in methylene chloride to give 0.443 g(80%) of pure product. H¹ - and C¹³ -Nuclear magnetic resonance spectrawere good.

The t-boc (0.10 g) and 10 ml of trifluoroacetic acid were allowed tostand at room temperature for 5 minutes. The excess acid was evaporatedand the residue triturated three times with ether. The ether was driedand concentrated to give crystals ofN-carbobenzoxy-L-alanyl-D,L-2-(3-aminopropylthio)-glycine, benzyl esteras the trifluoro acetic acid salt.

Anal. Calc'd for C₂₅ H₃₀ F₃ N₃ O₇.1.5H₂ O: C, 52.39; H, 5.36; N, 7.20.Found: C, 52.84; H, 5.85; N, 7.39.

This product exhibits the biological properties of a lysine-mimeticdipeptide. When exposed to biological fluid containing trypsin, thebenzyl ester is cleaved, then, carboxypeptidase B splits the peptidebond releasing --S(CH₂)₃ NH₂ which is detected using Ellman's reagent asdescribed hereinafter.

The homologous compound,N-carbobenzoxy-L-alanyl-D,L-2-(2-aminoethylthio)-glycine, benzyl etheris prepared using aminoethylthiol as starting material in the processdescribed above. Using 3-N-tosylguanidinopropylthiol, prepared as knownto the art, givesN-carbobenzoxy-L-alanyl-D,L-α-3-(N-tosylguanidinopropylthio)-glycine,benzyl ether which as an arginine-mimetic is useful as a chromogenicdetector of thrombin or thrombin derivatives in biological samples.

EXAMPLE 24

Substituting a stoichiometric quantity ofN-carbobenzoxy-L-phenylglycylamide, N-carbobenzoxy-L-phenyl-alanylamideor N,N-bis-carbobenzoxy-L-lysylamide in the reactions of Examples 1 and2 gives N-carbobenzoxy-L-phenylglycyl-2-hydroxy-D,L-glycine,N-carbobenzoxy-L-phenylalanyl-2-hydroxy-D,L-glycine andN,N-biscarbobenzoxy-L-lysyl-2-hydroxy-D,L-glycine.

N-carbobenzoxy-L-phenylglycyl-2-hydroxy-D,L-glycine is reacted with astoichiometric quantity of benzoyl chloride in pyridine to give the2-benzoyloxy compound which is treated with 5-fluorouracil and deblockedusing palladium-on-charcoal and cyclohexene as described in Example 2 togive L-phenylglycyl-2-(5-fluorouracil-1-yl)-D,L-glycine.

N-carbobenzoxy-L-phenylalanyl-2-hydroxy-DL-glycine is reacted withethylchloroformate in pyridine to give the 2-ethylformate which isreacted with 5-fluorouracil and deblocked to giveL-phenylalanyl-2-(5-fluorouracil-2-yl)-D,L-glycine.

N,N-biscarbobenzoxy-L-lysyl-2-hydroxy-D,L-glycine is acylated withacetyl chloride and pyridine, reacted with 5-fluorouracil and deblockedas described in Example 2 to giveL-lysyl-2-(5-fluorouracil-2-yl)-D,L-glycine.

EXAMPLE 25

Substituting N-carbobenzoxyleucine for N-carbobenzoxyalanine in Example5 gives D,L-leucyl-L-alanyl-2-(5-fluorouracil-1-yl)-D,L-glycine.Substituting N-carbobenzoxy-D,L-phenyl alanine givesD,L-phenylalanyl-L-alanyl-2-(5-fluorouracil-1-yl)-D,L-glycine.Substituting N-carbobenzoxy-D,L-alanyl-D,L-leucyl givesD,L-alanyl-D,L-leucyl-L-alanyl-2-(5-fluorouracil-1-yl)-D,L-glycine.Substituting N-carbobenzoxy-alanyl-alanyl-alanyl givesalanyl-alanyl-alanyl-L-alanyl-2-(5-fluorouracil-1-yl)-D,L-glycine.

Using either other amino acids or oligopeptides of the art, such asthose described in one of the volumes of "Synthetic Peptides", ofreference above, or other biologically useful warheads (W), othercompounds of this invention are prepared.

EXAMPLE 26

A mixture of 5 g (0.0266 m) of tert.-butoxycarbonyl alanyl amide, 3.9 g(0.03 m) of tert.-butyl glyoxylate, prepared by oxidation of tert.-butyltartrate, and 100 ml of benzene is heated at reflux using azeotropicconditions for 54 hours to complete the reaction to giveN-tert.-butoxy-carbonyl-L-alanyl-2-hydroxy-D,L-glycine, tert.-butylester which is used as above with appropriate removal of the t.-bocgroups such as reacting with trifluoroacetic acid.

Antimicrobial Examples

A prototypal compound, L-alanyl-2-(5-fluorouracil-2-yl)-L-glycine, wasevaluated against representative fungal and bacterial organisms.

Methods of Testing

A. Fungi (Disk diffusion method):

Candida albicans strains B-311 and BC 759, obtained from Smith Kline andFrench Laboratory Culture Collection were grown in a trypticase soybroth at 28° for 7 hours. The seeded plates, with this medium containing1.5% agar, were prepared by inoculating 1 ml of the inoculum in 1 l. ofyeast-carbon base medium, containing 200 μg/ml of lysine or glutamate asa sole source of nitrogen.

B. Fungi [Minimum inhibitory contraction (MIC)]:

The minimum inhibitory concentration of the test compound was determinedby two-fold broth dilution tests in yeast carbon base medium, containing200 μg/ml of sodium glutamate. The compounds were diluted from 280 μg/mlto 0.25 μg/ml and the test medium was inoculated with a suspension of C.albicans grown in the same medium for 15 hours at 37°. The finalinoculum sizes in the tests were approximately 10⁴ cfu/ml and 10³cfu/ml. The tubes were incubated at 37° for 24 and 48 hours and observedfor inhibition. C. Bacteria (Disk diffusion method):

A method similar to A above but using E. coli was used which employs aM-9 medium (basic salts with ammonium ion as the nitrogen source andglucose at the carbon source).

Results of Testing

(1) C. albicans in Method A:

    ______________________________________                                        L-alanyl-2-(5-fluorouracil-                                                                     L-alanyl-2-(5-fluorouracil-                                 1-yl)-L-glycine   1-yl)-D-glycine                                             μg/ml                                                                             zone size (mm) μg/ml zone size (mm)                                 ______________________________________                                        1000   30             1000     0                                              500    26             500      0                                              250    22             250      0                                              125    20             125      0                                               62    16              62      0                                              ______________________________________                                    

These data demonstrate the unique contribution of the L-L configurationin the compounds of this invention.

(2) C. albicans in Method B:

    ______________________________________                                        Alanyl-2-(5-fluorouracil-2-yl)-glycine                                        MIC (μg/ml)                                                                Strains                                                                              5-FU   L-DL      L-L  L-D     Amphotericin B                           ______________________________________                                        BC 759 1.0    6.3       1.6  >100    1.25                                     B 311  4.0    25        12.5 >100    1.25                                     3153A  1.0    25        12.5 >100    1.25                                     ______________________________________                                    

These data indicate antifungal activity for the L- DL and, especially,the L-L-diastereoisomers but none for the L-D isomer.

(3) E. coli M. 2626 in Method C:

    ______________________________________                                                                   2-FU-L-                                                          Zone size (mm)                                                                             alanyl-L-                                                              FU + 2-FU- FU +  glycine +                                          5-fluoro  alanyl-L-  alanyl-                                                                             alanyl-                                  conc. (μg/disk)                                                                      uracil (FU)                                                                             glycine    alanine                                                                             alanine                                  ______________________________________                                        25.0      46        31         --    --                                       12.5      41        26         41    0                                        6.0       40        27         40    0                                        3.0       38        25         38    0                                        1.5       33        22         33    0                                        ______________________________________                                    

These data show activity is due to prodrug itself, not to itspre-dissociated parts.

(4) C. albicans B-310 in Test B:

    ______________________________________                                        MIC (μg/ml, 24 hours                                                       L-alanyl-2-(5-fluorouracil-                                                                    L-alanyl-2-(5-fluorouracil-                                  2-yl)-L-glycine  2-yl)-D-glycine                                              ______________________________________                                        12.5             >200                                                         ______________________________________                                    

(5) C. albicans B-311 in Test A:

    ______________________________________                                        Alanyl-2-(5-fluorouracil-2-yl)-glycine                                        Conc. (μg/desk)                                                                          5-Fu   L-DL       L-L  L,D                                      ______________________________________                                        25            44     20         30   0                                        12.5          40     20         26   0                                        6.0           30     20         22   0                                        3.0           30     18         20   0                                        1.5           22     12         16   0                                        ______________________________________                                    

These data confirm the activity of the isomer with a L-configuration inthe terminal glycine unit of the oligopeptide chain.

(6) Stability of α-substituted Glycine Peptides to Aqueous Solutions andEnzyme Catalyzed Warhead Release:

The α-substituted glycine peptide of Example 4, having a structure whichcontains a sulfhydryl group linked at the α-position of glycine, wasfound to be stable (t 1/2˜5000 min) in 0.2M phosphate buffer (pH 7.0).Upon addition of the enzyme, Leucine Aminopeptidase, the warhead wasreleased. The release was maintained at a linear rate over theobservation period (5 min). This observation was repeated at pH 7.5, atwhich pH there was, again, no noticeable increase in the non-enzymaticwarhead release; however, the enzymatically catalyzed release againproceeded at twice the previous rate. This observation is consistentwith literature reports of the optimal pH necessary for the efficacy ofthis enzyme. Similar results were obtained with the product of Example21.

The procedure described here is applied to any enzyme which is capableof hydrolyzing the stabilizing peptide bond, such as that between units1 and 2 of the enzyme substrates of formula 1, for example, enzymes suchas carboxypeptidase A or trypsin or the various serum proteases. Theprocedure described here is a more versatile detection system than thoseused in the prior art which use chromogenic peptide substrates such asleucine p-nitroanilide.

These observations were obtained by colorimetric assay, which involvesthe reaction between a free sulfhydryl group and5,5'-dithio-bis(2-nitrobenzoic acid), the Ellman reagent, to produce ayellow color as follows: ##STR12## Assay:

The peptide from Example 4 was dissolved in 4 mM of phosphate buffersolution (pH 7) containing the Ellman reagent at a level of 4 mg/ml toproduce a 1 mM solution of the peptide. The resulting mixture wasmonitored using a spectrophotometer at a wavelength of 412 nm. To 1 mlof this solution was added 100 μg of hog kidney leucine aminopeptidase(Sigma) and the enzymatic hydrolysis was continuously monitored at 412nm.

    ______________________________________                                        Data                                                                                         Phosphate buffer                                               Time (sec)     λ(412)                                                  ______________________________________                                        0              0.090                                                          1              0.093                                                          2              0.095                                                          enzyme added:                                                                 3              0.109                                                          4              0.127                                                          5              0.142                                                          6              0.161                                                          7              0.180                                                          8              0.199                                                          ______________________________________                                    

Reaction mixture contained 1 mM peptide, Ellman's reagent 4 mg/ml,phosphate buffer 4 mM (pH 7).

    ______________________________________                                                      p moles per min                                                 ______________________________________                                        Spontaneous Rate                                                                               65                                                           plus 100 μg enzyme                                                                         504                                                           ______________________________________                                    

What is claimed is:
 1. A compound of the structural formula: ##STR13##in which: Y is a carboxyl protective group;R¹ is a C₁₋₄ lower alkyl,phenyl, Z--NHC₃ H₆ --, or benzyl; R⁵ is chloro, bromo, hydroxy oracyloxy; and Z is an amino-protective group.
 2. The compound of claim 1in which R¹ is methyl.
 3. The compound of claim 1 in which Y is benzyland R¹ is methyl and Z is carbobenzoxy.
 4. The compound of claim 1 inwhich R⁵ is acetoxy.
 5. The compound of claim 1 in which Y is allyl andR¹ is methyl and Z is allyloxycarbonyl.
 6. The compound of claim 1 beingN-allyloxy-carbonyl-L-alanyl-2-acetoxy-D,L-glycine, allyl ester.
 7. Thecompound of claim 1 beingN-allyloxy-carbonyl-L-alanyl-2-hydroxy-D,L-glycine, allyl ester.
 8. Thecompound of claim 1 being N-carbobenzoxy-L-alanyl-2-acetoxy-D,L-glycine,benzyl ester.
 9. The compound of claim 1 beingN-carbobenzoxy-L-alanyl-2-hydroxy-D,L-glycine, benzyl ester.
 10. Thecompound of claim 1 beingN-tert.-butyl-oxycarbonyl-L-alanyl-2-acetoxy-D,L-glycine, tert.-butylester.